RELM beta pre-activated stem cell outer vesicle with high anti-inflammatory and anti-fibrosis activity

Abstract

The invention belongs to the technical field of stem cell culture and exosome extraction, and provides an RELM beta pre-activated stem cell outer vesicle with high anti-inflammatory and anti-fibrosis activity. According to the method, the mesenchymal stem cells are subjected to hunger culture after being adhered to the wall and then are continuously cultured by a complete culture medium containing RELM beta factors, then supernate is collected, and the exovesicles are obtained through separation. In the preparation process of the extracellular vesicles, the paracrine function of MSC cells is promoted by the RELM beta, the secretion of anti-inflammatory and anti-fibrosis active factors is obviously activated, and the generation of the extracellular vesicles with enhanced anti-inflammatory and anti-fibrosis effects is promoted. Gene modification is not involved, toxic biochemical reagents or materials are not added, exogenous operation steps are simple and convenient, clinical application is guaranteed through sterile operation, no RELM beta residues exist after extracellular vesicle extraction, and the method is safe, non-toxic and effective.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell culture and exosome extraction, and specifically relates to the preparation and application of anti-inflammatory and anti-fibrotic stem cell exosomes. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Mesenchymal stem cells (MSCs) are derived from the mesoderm and have the function of repairing tissue damage. Extracellular vesicles (EVs) are vesicle-like substances secreted by cells, which have similar functions to the source cells. In recent years, mesenchymal stem cell-derived EVs (MSC-EVs) have received extensive attention and have played a therapeutic role in various organ diseases such as the lungs, intestines, and nervous system. Recent studies have found that the immunoregulatory function of MSCs in a resting state is very weak and the therapeutic effect is poor; however, after being stimulated and activated by inflammatory factors such as IFN-γ, TNF-α, IL-1β, and NO, MSCs can fully exert their immunoregulatory function. In order to further improve the therapeutic effect of stem cell EVs, different pathophysiological conditions and stimulation of parental cells are known to change the content of exosomes and may regulate the function of exosomes. Enhancement strategies, including genetic manipulation, pre-activation, and changes in culture methods, have been studied to produce highly functional MSCs. Compared with genetic manipulation, pre-activation overcomes the safety issues of transgenics and is an alternative and feasible method to improve the therapeutic outcomes of MSCs. Pre-activation is simulated by pathological microenvironments to achieve the desired functions and reverse their inactivation. Fine-tune the characteristics of cells in response to harsh environments to make them suitable for target diseases. Based on the plasticity and memory ability of MSCs, signals in the traumatic microenvironment, such as environmental factors (hypoxia) and chemical factors (inflammatory factors and cytokines), will be the primary consideration for the in vitro operation of MSCs. For example, TNF-α enhances the in vitro hematopoietic support function of human umbilical cord mesenchymal stem cell conditioned medium; IL-1β combined with TNF-α pretreatment of bone marrow mesenchymal stem cells promotes the expression of vascular cell adhesion molecule-1 and improves post-ischemic cardiac function.

[0004] Resistin-like molecule beta (RELM β) is an inflammatory factor involved in a variety of chronic diseases discovered in recent years, and is also known as FIZZ2 (found in inflammatory zone 2). The resistin-like molecule family (RELMs) is a cysteine-rich family newly discovered by Holcomb et al. in the bronchoalveolar lavage fluid of mice with asthma in 2000. Studies have shown that RELM is widely involved in hypoxia and downstream inflammatory signals, and mainly plays a role in the lungs, blood vessels and intestines. Clinical studies have shown that increased expression of RELM β was found in the lungs of patients with COPD, pulmonary fibrosis, pulmonary hypertension and asthma. Further studies have shown that RELM β, as an inflammatory mediator specifically expressed in lung tissue, is upregulated in inflammatory cells, promotes fibroblast proliferation and differentiation, and the deposition of extracellular matrix proteins, leading to the occurrence and development of a variety of lung diseases. For example, hypoxia can upregulate the expression of RELMβ in human pulmonary artery smooth muscle cells, promote the proliferation of pulmonary artery smooth muscle cells, and then lead to pulmonary hypertension; in the airway inflammation response of asthma patients, RELMβ may be induced by Th2 cytokines, affecting the occurrence and development of asthma, promoting the proliferation of airway epithelium and smooth muscle, mucus secretion, collagen fiber, fibronectin synthesis, and strengthening the connection between hyaluronic acid and proteoglycan protein 1, leading to airway inflammation and remodeling; RELMβ causes pulmonary fibrosis by increasing pulmonary fibrosis protein. In addition, RELMβ can regulate the integrity of the colon barrier and susceptibility to inflammation. RELM, as a newly discovered protein family, mediates the activation of multiple signal transduction pathways as an initiating regulatory factor, and plays a role in pro-inflammatory, pro-proliferative, pro-growth, pro-secretion, chemotaxis and immunomodulatory effects. At present, there is no report on inducing umbilical cord mesenchymal stem cells through RELMβ to improve their anti-inflammatory and anti-fibrotic ability to secrete Ev. Summary of the invention

[0005] In order to obtain stem cell extracellular vesicles with better anti-inflammatory and anti-fibrosis abilities, the present invention provides a method for preparing extracellular vesicles, inducing umbilical cord mesenchymal stem cells by RELM β, and then separating and obtaining extracellular vesicles, which have better anti-inflammatory and anti-fibrosis abilities.

[0006] Another object of the present invention is to provide the use of the above-mentioned extracellular vesicles in the treatment of idiopathic pulmonary fibrosis and asthma.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A method for preparing stem cell extracellular vesicles comprises the following steps: (1) Starvation culture of mesenchymal stem cells after adherence; (2) replacing the mesenchymal stem cells in step (1) with complete culture medium containing RELM β factor and continuing the culture; (3) Collect the supernatant from step (2) and separate the extracellular vesicles.

[0009] In step (1), the culture medium used before adhesion is a complete culture medium; the culture medium for starvation culture is a serum-free culture medium, and the culture time is 1h-4h. The serum-free culture medium refers to a complete culture medium without serum or serum substitute. In order to exclude the influence of serum in the culture medium, the serum in the culture medium is a serum substitute or serum without extracellular vesicles.

[0010] In step (2), the concentration of RELM β factor in the culture medium is 5-50 ng / mL.

[0011] The source of mesenchymal stem cells is not limited, such as the source of the stem cells mentioned above, which can be placental tissue, umbilical cord blood, bone marrow, fat, dental pulp, urine, menstrual blood, etc. In order to maintain a high activity, the mesenchymal stem cells can be selected from 3 to 10 generations.

[0012] The method for separating the extracellular vesicles can adopt methods in the prior art, including but not limited to ultrafiltration, tangential flow ultrafiltration, ultrahigh speed centrifugation, density gradient centrifugation, magnetic bead immunoseparation, size exclusion chromatography and the like.

[0013] The stem cell extracellular vesicles can be used to prepare anti-allergic, anti-inflammatory and anti-fibrotic drugs; the drugs are mainly used to treat lung diseases; the lung diseases are selected from idiopathic pulmonary fibrosis and asthma.

[0014] The administration form of the drug is not particularly limited, and is preferably mucosal administration, such as aerosolization.

[0015] The present invention has the following advantages: (1) The present invention promotes the paracrine function of MSC cells by pre-activating the factor RELM β, significantly activates the secretion of anti-inflammatory and anti-fibrotic active factors, and promotes the production of extracellular vesicles with enhanced anti-inflammatory and anti-fibrotic effects; (2) Compared with the existing technology, the present invention does not involve gene modification, does not involve the addition of toxic biochemical reagents or materials, has simple exogenous operation steps, and aseptic operation ensures clinical application. There is no RELMβ residue after extracellular vesicle extraction, which is safe, non-toxic and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a comparison of the secretion of anti-inflammatory and anti-fibrotic factors by conventional culture and pre-activated MSCs, including: A: detection of IL-10 content; B: detection of Arg-1 content; C: detection of MMP-9 content, p<0.05; p<0.001; p<0.0001; Figure 2 This is a comparison of the anti-inflammatory effects of secretory components of conventionally cultured and pre-activated MSCs. vs blank p<0.001; # vs BLM+PBS p<0.05; ## vs BLM+PBS p<0.01; $ vs BLM+Ev, p<0.05; Figure 3 The expression level of α-SMA in TGF-β1-induced HSF cells by conventional culture and pre-activated MSC secretory components. vs blank p<0.0001, # vs TGF-β1+PBS p<0.05, ## vs TGF-β1+PBS p<0.01; ### vs TGF-β1+PBS p<0.001; $ vs TGF-β1+Ev p<0.05; Figure 4 The effects of conventional culture and pre-activated MSC secretion of EV on the levels of TNF-α (A), IL-6 (B), and IL-1β (C) in bronchoalveolar lavage fluid of IPF mice. vs blank p<0.01, vs blank p<0.001, # vs BLM+PBS p<0.05; ##vs BLM+PBS p<0.01, $ vs BLM-β1+Ev p<0.05; Figure 5 The effect of conventional culture and pre-activated MSC secretion of Ev on the content of α-SMA in the lung tissue of IPF mice, where A: WB detection, B: WB data quantitative analysis bar graph, vs blank p<0.001, ## vs BLM+PBS p<0.01; ###vs BLM+PBS p<0.001, $ vs BLM-β1+Ev p<0.05; Figure 6 It is the serum IgE ELISA test of asthma model mice. vs blank p<0.0001, ## vs OVA+PBS p<0.01; ### vs OVA+PBS p<0.001, $ vs OVA+Ev p<0.05; Figure 7 is the content of inflammatory cells in the bronchoalveolar lavage fluid of asthma model mice, among which A: neutrophils, B: eosinophils, vs blank, vs blank p<0.0001, ## vs OVA+PBS p<0.01, ### vs OVA+PBS p<0.001, $$ vs OVA+Ev p<0.01. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0018] Example 1 Preparation of stem cell extracellular vesicles 1. Pre-stimulation of Mesenchymal Stem Cells P1 MSCs were taken out of liquid nitrogen and quickly thawed in a 37°C water bath. The cells were transferred to 10 mL of complete medium and centrifuged at 300 g for 5 min. The cells were resuspended in complete medium and counted. The number of cells was 15,000 cells / cm 2 Inoculate one T75 flask, add complete medium and continue to culture for 3 days; when the cell confluence is greater than 90%, digest the P2 cells with Tryple and subculture them to 3 T75 flasks at a ratio of 1:3, where the medium in the T75 flasks is still complete medium, and check the confluence of P3 MSCs after continuing to culture for 3 days; when the cell confluence is greater than 90%, digest the P3 MSCs with Tryple and expand them to 9 T175 flasks containing complete medium at a ratio of 1:3 and continue to culture; when the P4 MSC confluence is greater than 90%, digest the P4 MSCs and resuspend the precipitate, count them with a cell counter, and count every 1.3×10 7 Add 100 cells to a 250 mL centrifuge tube, add complete medium to the volume to 150 mL, mix the cells evenly, transfer to a 5-layer flask for culture, and then perform pre-stimulation according to the following steps: (1) After the P5 MSCs were cultured until the cells adhered to the wall, the 5-layer flask was washed three times with PBS preheated at 37°C, and the serum-free culture medium was replaced. The flask was starved and cultured at 37°C for 2 hours. (2) Recombinant human RELM β was diluted to a concentration of 100 μg / mL with 0.1% serum replacement and filtered through a 0.22 μm filter to obtain the mother solution; the recombinant human RELM β mother solution was diluted to 50 ng / mL with serum-free medium and replaced with the serum-free medium in (1), and P5 MSCs were cultured at 37°C for 48 h; (3) The culture supernatant was collected and the content of anti-inflammatory and anti-fibrotic factors IL-10, Arg-1 and MMP-9 in the culture supernatant was detected using an ELISA kit, with MSCs not stimulated with recombinant human RELMβ as a control.

[0019] The results are as follows Figure 1As shown in Figure 2, compared with MSCs cultured under the same culture conditions without pre-activation, MSCs pre-activated by recombinant human RELMβ secreted more IL-10 ( vs MSC supernatant), Arg-1 ( vs MSC supernatant) and MMP-9 ( vs MSC supernatant), and was significant.

[0020] 2. Extracellular vesicles (RELM-β-MSC-Ev) isolation The culture supernatant of the MSCs pretreated with the above recombinant human RELM β was collected, and the extracellular vesicles were isolated according to the following steps: (1) Centrifuge at 4°C, 300 g for 10 min and collect the supernatant; centrifuge at 4°C, 2000 g for 10 min and collect the supernatant; centrifuge at 4°C, 10000 g for 30 min and collect the supernatant; (2) Take the supernatant for tangential flow concentration: The purification column used for tangential flow is a 100 KDa hollow fiber column. Reduce the pump speed to below 300 mL / min and run the pump. After concentrating 10 L of liquid to 200 mL, collect the concentrate; (3) Add the concentrate to an ultracentrifuge tube and ultracentrifuge at 120,000 g at 4°C for 120 min. Collect the precipitate, wash once with sterile PBS, and continue ultracentrifugation at 120,000 g at 4°C for 90 min. (4) Slowly discard the supernatant, resuspend the precipitate with medical saline, mix thoroughly, add the liquid to the ultracentrifuge tube, and centrifuge at 10,000 g at 4°C for 30 min; (5) Take the supernatant, which is the extracellular vesicle (RELM-β-MSC-Ev) suspension.

[0021] 3. In vitro anti-inflammatory effect The supernatant obtained in 1, the concentrate obtained in 2 (2), and the extracellular vesicles obtained in 2 were subjected to in vitro anti-inflammatory experiments: After digestion, the MLE-12 cells were counted and the complete medium was prepared to a cell density of 2.5×10 5 100 g / mL of cell suspension, mix well, and spread 2 mL of cell suspension into a 6-well plate; place the 6-well plate in a cell culture incubator and culture at 37°C for 24 hours; replace with fresh culture medium, and add bleomycin and the substance to be tested according to the grouping: The blank control group, bleomycin induction + PBS group, bleomycin + supernatant group, bleomycin + concentrate group, and bleomycin + extracellular vesicle group were set up; the final concentration of bleomycin was 100 μg / mL, and the final concentration of Ev group was 5×10 9 Number of particles / mL, final concentration of supernatant and concentrated supernatant 20% (v / v); After mixing, the cells were cultured for 24 h, and the culture supernatant was collected for ELISA detection. The OD value was measured at 450 nm.

[0022] Depend on Figure 2 It can be seen that bleomycin (BLM) induces MLE-12 cells to produce a large amount of inflammatory factor TNF-α ( , p<0.01), the culture supernatant and concentrate of MSC under conventional culture did not significantly inhibit the TNF-α induced by BLM, which is consistent with most reports that the EV secreted by stem cells has the effect of inhibiting TNF-α. Compared with BLM+PBS, the supernatant and concentrate secreted by pre-activated MSC have better anti-inflammatory ability, and the effect is roughly consistent with the EV secreted by MSC (#, p<0.05). The best effect is the EV secreted by pre-activated MSC, which has a higher anti-inflammatory ability than all the tested substances (##, p<0.01).

[0023] 4. In vitro anti-fibrotic effect The supernatant obtained in 1, the concentrate obtained in 2 (2), and the extracellular vesicles obtained in 2 were subjected to in vitro anti-fibrosis experiments: HSF cells were seeded into six-well plates and cultured at 37°C for 24 h. Fresh culture medium was then replaced and TGF-β1 and the substances to be tested were added according to the groups: The blank control group, TGF-β1 induction + PBS group, TGF-β1 + supernatant group, TGF-β1 + concentrate group, and TGF-β1 + extracellular vesicle group were set up; the final concentration of TGF-β1 was 10 ng / mL, and the final concentration of Ev group was 5×10 9 Number of particles / mL, final concentration of supernatant and concentrated supernatant 20% (v / v); After mixing, the cells were cultured for another 24 h, the culture medium was discarded, the cells were digested with trypsin, and the cells were collected and RNA was extracted using an RNA kit to detect the gene expression of α-SMA.

[0024] The results are as follows Figure 3 As shown: In the HSF cell model treated with TGF-β, the gene expression of fibrosis marker protein α-SMA was increased ( p<0.0001). The concentrated supernatant and Ev of conventionally cultured MSCs both have the ability to inhibit fibrosis. The culture supernatant, concentrated solution and Ev of pre-activated MSCs have better anti-fibrosis effects than the unactivated experimental substances of the same source. Data analysis showed that the anti-fibrosis ability of pre-activated Ev was further enhanced compared with Ev isolated from conventionally cultured MSCs ($p<0.05).

[0025] Application Example 1 Application of RELM-β-MSC-Ev in the treatment of idiopathic pulmonary fibrosis (IPF) 1. Modeling The mice were weighed before the experiment and divided into a normal group, a model group, a MSC-Ev treatment group, and a RELM-β-MSC-Ev treatment group (referred to as R-MSC-Ev). Before modeling, the mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital. After successful anesthesia, the mice were fixed on the operating table, the tongue of the mice was held by hand and pulled to the lower left to expose the glottis, and the catheter was inserted into the mouse trachea. Under the condition of ensuring normal breathing of the mice, 50 μL of BLM was dripped into each mouse in each dose group, with a final concentration of 8 U / kg. After the liquid was completely inhaled into the trachea, the catheter was quickly pulled out and the mice were upright. The mice in the normal group were not dripped with BLM.

[0026] 2. Treatment A Yuyue 403M nebulizer (nebulization rate of 0.2 mL / min) was used for nebulization. The Ev concentration was 1×10 10 Particle number / mL, nebulization twice a day, nebulization for 30 minutes each time, for 7 consecutive days; the control group nebulized normal saline.

[0027] 3. Bronchoalveolar lavage On the second day after treatment, mice were killed by cervical dislocation / anesthesia, fixed on the operating table, and the neck was sprayed with alcohol to expose the trachea. The surgical line was passed under the trachea, a small incision was made above the trachea closest to the mouth, and a tracheal tube was inserted. The alveolar lavage fluid was repeatedly aspirated with 1 mL PBS. The collected alveolar lavage fluid was centrifuged at 500 g for 5 min, and the supernatant was collected for ELISA detection of inflammatory factors. The content of IL-6, IL-1β, and TNF-α in the supernatant of the alveolar lavage fluid was detected using the PTG Elisa kit.

[0028] like Figure 4 As shown in the figure, the expression of inflammatory factors TNF-α, IL-6 and IL-1β in the bronchoalveolar lavage fluid of BLM-induced IPF mice increased, which was consistent with the characteristics of the model, indicating that the modeling was successful. Compared with PBS treatment, MSC-Ev treatment can significantly reduce the expression of the above inflammatory factors. Compared with MSC-Ev treatment, Ev obtained by pre-activating MSC has a better anti-inflammatory effect (p < 0.05).

[0029] 4. WB detection of α-SMA content in mouse lung tissue (1) After 28 days of treatment, 20-30 mg of lung tissue was collected, cut into small pieces, added with 200 μL RIPA lysis buffer, and fully lysed using a low-temperature high-speed grinder. After grinding, place on ice for 20 min. Centrifuge at 12,000 rpm and 4°C for 15 min, take the supernatant, and measure the BCA protein concentration.

[0030] (2) Load the protein to be tested into the protein spotting hole of the polyacrylamide gel, adjust the voltage to 80 V, and run for 20 min. Then set 100 V until bromophenol blue reaches the bottom of the separation gel. Transfer the protein to the PVDF membrane at 200 mA for 100 min. Block with 5% skim milk powder at 4°C overnight, add α-SMA and α-tubulin primary antibodies, and incubate at 4°C overnight. Use the secondary antibody for color development, and use GraphPad Prism 8.4.0 data analysis and plotting software to analyze and process the data. All data are expressed as Mean±SD.

[0031] like Figure 5 As shown in Figure 2, BLM induction caused the formation of pulmonary fibrosis in IPF mice on day 28, and the expression of fibrosis marker protein α-SMA in lung tissue was significantly increased ( p<0.001). After treatment with conventional MSC-Ev or pretreated MSC-Ev, the pulmonary fibrosis of IPF mice was alleviated and the expression of α-SMA was significantly decreased. Among them, Ev obtained by pre-activated MSC had a better anti-fibrotic therapeutic effect (p<0.05).

[0032] Application Example 2 Application of RELM-β-MSC-Ev in the treatment of asthma 1. Modeling BALB / c mice were randomly divided into normal control group (NC group) and nasal drop provocation group after 7 days of adaptation to the new environment. The day of modeling was recorded as the 0th day of the experiment. On the 0th, 7th and 14th days of the experiment, each mouse was intraperitoneally injected with 500 μL of sensitizer for sensitization (100 μg OVA per mouse). On the 21st day of modeling, OVA nasal drops were used to stimulate nasal drops once a day for 3 consecutive days. After the provocation, the mice were divided into control group, OVA+PBS, OVA+MSC-Ev and OVA+R-MSC-Ev groups. A Yuyue 403M nebulizer (nebulization rate of 0.2 mL / min) was used for nebulization. The Ev concentration was 1×10 10 The number of particles / mL was measured, and the patients were nebulized twice a day, each time for 30 minutes, for 7 consecutive days; the control group was nebulized with normal saline for 5 consecutive days.

[0033] After the nebulization, blood was collected from the mouse orbits, and serum was collected for Elisa IgE testing. After the mouse bronchoalveolar lavage fluid was drawn, the number of eosinophils and neutrophils was detected using a small animal blood cell analyzer.

[0034] like Figure 6As shown in the figure, the OVA-specific IgE test results showed that the model group (OVA+PBS) was strongly positive compared with the control group. MSC-Ev and pre-activated MSC-secreted Ev both inhibited the allergic reaction of asthmatic mice and significantly reduced the level of IgE in serum. Among them, compared with MSC-Ev, the Ev secreted by pre-activated MSC had a stronger anti-allergic effect ($p<0.05).

[0035] Asthma is a chronic inflammatory disease of the airways with multiple inflammatory patterns; among them, eosinophils (EOS) are important inflammatory cells in airway inflammation. EOS destroys the integrity of airway epithelial cells, causing them to be damaged and fall off, inducing airway inflammation and airway hyperresponsiveness. If not controlled for a long time, it will cause airway remodeling. Figure 7 As shown, in OVA-induced allergic asthma mice, neutrophils ( p<0.0001) and eosinophils ( MSC-Ev and Ev secreted by pre-treated MSC can significantly inhibit the production of neutrophils and eosinophils in allergic asthma. Compared with MSC-Ev, Ev secreted by pre-treated MSC has a more significant anti-inflammatory effect ($$p<0.01).

[0036] The above results show that the secretory supernatant of the pretreated activated MSC provided by the present invention contains more anti-inflammatory molecules Arg-1 and IL-10, which can significantly inhibit the release of inflammatory factors of lung epithelial cells MLE-12 caused by bleomycin compared with the unpretreated MSC culture supernatant; inhibit TGF-β1-induced MLE-12 cell fibrosis. At the same time, the extracellular vesicles extracted from the culture fluid effectively inhibited the inflammatory factors in the alveolar lavage fluid (BALF) and the formation of fibrosis in the lung tissue when treating IPF mice; when treating asthmatic mice, it effectively inhibited the allergic reaction and airway hyperreaction of mice and reduced the serum IgE content. The method provided by the present invention can effectively increase the level of anti-inflammatory and anti-fibrotic factors secreted by umbilical cord mesenchymal stem cells, and further enhance the above functions of extracellular vesicles.

[0037] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for preparing stem cell extracellular vesicles, characterized in that: The following steps are involved: (1) Starvation culture of mesenchymal stem cells after adherence; (2) replacing the mesenchymal stem cells in step (1) with complete culture medium containing RELM β factor and continuing the culture; (3) Collect the supernatant from step (2) and separate the extracellular vesicles.

2. The preparation method according to claim 1, characterized in that: In step (1), the culture medium used before adhesion is a complete culture medium; the culture medium for starvation culture is a serum-free culture medium, and the culture time is 1 hour to 4 hours; the serum-free culture medium is a complete culture medium that has been free of serum or serum substitute.

3. The preparation method according to claim 2, characterized in that: In step (1), the serum in the complete culture medium is a serum replacement or serum without extracellular vesicles.

4. The preparation method according to claim 1, characterized in that: In step (2), the concentration of RELM β factor in the culture medium is 5-50 ng / mL.

5. The preparation method according to claim 1, characterized in that: The source of mesenchymal stem cells is selected from placental tissue, umbilical cord blood, bone marrow, fat, dental pulp, urine or menstrual blood; the passage number of mesenchymal stem cells is 3-10.

6. The preparation method according to claim 1, characterized in that: The method for isolating the extracellular vesicles is selected from ultrafiltration, tangential flow ultrafiltration, ultracentrifugation, density gradient centrifugation, magnetic bead immunoseparation or size exclusion chromatography.

7. A stem cell extracellular vesicle obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the stem cell extracellular vesicles as claimed in claim 7 in the preparation of anti-allergic, anti-inflammatory and anti-fibrosis drugs.

9. The use according to claim 8, characterized in that: The drug is used for treating lung diseases; the lung diseases are selected from idiopathic pulmonary fibrosis and asthma; and the drug is preferably in a mucosal administration dosage form.

10. A medicine prepared by the stem cell extracellular vesicles as claimed in claim 7.

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